aromatic amino acids in the brain...in normal brain function, in the pathophysiology of various...

Aromatic Amino Acids in the Brain

Ciba Foundation Symposium 22 (new series)

1974

Elsevier . Excerpta Medica . North-Holland Associated Scientific Publishers . Amsterdam . London . New York

The Ciba Foundation for the promotion of international cooperation in medical and chemicul research is a scientific and educational charity established by CIBA Limited - now CIBA-GEIGY Limited - of Basle. The Foundation operates independently in London under English trust law.

Cibu Foundation Symposia are published in collaboration with Associated Scientific Publishers (Elsevier Scientific Publishing Company, Excerptu Medica, North- Hollmd Pit Slishing Company) in A msterdan? .

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1974

Elsevier . Excerpta Medica . North-Holland Associated Scientific Publishers . Amsterdam . London . New York

0 Copyright 1974 Ciba Foundation

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without permission in writing from the publishers.

ISBN Excerpta Medica 90 219 4023 x ISBN American Elsevier 0-444-1 5019-6

Library of Congress Catalog Card Number 73-91643

Published in 1974 by Associated Scientific Publishers, P.O. Box 211, Amsterdam, and American Elsevier, 52 Vanderbilt Avenue, New York, N.Y. 10017.

Suggested series entry for library catalogues: Ciba Foundation Symposia. Suggested publisher’s entry for library catalogues: Associated Scientific Publishers.


Printed in The Netherlands by Mouton & CO, The Hague

Contents

R. J. WURTMAN Chairman’s introduction I

H. N. MUNRO Control of plasma amino acid concentrations 5 Discussion 18

A . LAJTHA Amino acid transport in the brain ivl vivo and in vitro 25 Discussion 4 1

J. AXELROD and I. M. SAAVEDRA Octopamine, phenylethanolamine, phenyl- ethylamine and tryptamine in the brain Discussion 60

51

P. MANDEL and D. AUNIS Tyrosine aminotransferase in the brain 67 Discussion 79

s. KAUFMAN Properties of pterin-dependent aromatic amino acid hydroxyl- ases 85 Discussion 108

A. CARLSSON The in vivo estimation of rates of tryptophan and tyrosine hy- droxylation: effects of alterations in enzyme environment and neuronal activity 117 Discussion 126

N. WEINER, F.-L. LEE, J . c. WAYMIRE and M. POSIVIATA The regulation of tyrosine hydroxylase activity i n adrenergic nervous tissue Discussion 147

135

J. D. FERNSTROM, B. K. MADRAS, H. x. MUNRO and R. J. WURTMAN Nutritional control of the synthesis of 5-hydroxytryptamine in the brain 153 Discussion 166

VI CONTENTS

A. PARFITT and D. G. GRAHAME-SMITH The transfer of tryptophan across the synaptosome membrane 175 Discussion 192

A. T. B. MOIR Tryptophan concentration in the brain 197

G. L. GESSA and A. TAGLIAMONTE Serum free tryptophan: control of brain con- 207

G. CURZON and P. J. KNOTT Fatty acids and the disposition of tryptophan 217

centrations of tryptophan and of synthesis of 5-hydroxytryptamine

Discussion of the three preceding papers 230

M. BULAT Monoamine metabolites in the cerebrospinal fluid: indicators of the biochemical status of monoaminergic neurons in the cerebral nervous system 243 Discussion 257

s. H. BARONDES Do tryptophan concentrations limit protein synthesis at specific sites in the brain? 265 Discussion 275

s. s. OJA, P. LAHDESMAKI and M.-L. VAHVELAINEN Aromatic amino acid supply and protein synthesis in the brain 283 Discussion 294

S. ROBERTS Effects of amino acid imbalance on amino acid utilization, protein synthesis and polyribosome function in cerebral cortex 299 Discussion 3 18

B. F. WEISS, L. E. ROEL, H. N. MUNRO and R. J . WURTMAN L-Dopa, polysomal aggregation and cerebral synthesis of protein Discussion 332

325

L. MAhRE, P. R. HEDWALL and P. C. WALDMEIER a-Methyldopa, an unnatural aromatic amino acid 335

E. M. GAL Synthetic p-halogenophenylalanines and protein synthesis in the brain 343 Discussion 354

T. L. SOURKES Effects of a-methyltryptophan on tryptophan, 5-hydroxytryptamine and protein metabolism in the brain 361 Discussion 378

R. J. WURTMAN Chairman’s closing remarks 381

Index of contributors 385

Subject index 387

Participants

Symposium on Aromatic Amino Acids in the Brain held at the Ciba Foundation on 15-17th May, 1973

R. J. WURTMAN (Chairman) Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 021 39, USA

Research Program, National Institute of Mental Health, 9000 Rockville Pike, Bethesda, Maryland 20014, USA

s. H. BARONDES Department of Psychiatry, School of Medicine, University of California San Diego, PO Box 109, La Jolla, California 92037, USA

M. BULAT Department of Pharmacology, Chicago Medical School, 2020 West Ogden Avenue, Chicago, Illinois 60612, USA

A. CARLSSON Department of Pharmacology, University of Goteborg, Fack, S-400 33 Goteborg 33, Sweden

G. CURZON Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WIC 3BG

1 George Square, Edinburgh EH8 9JZ

Strasse 1 , CH-8008 Zurich, Switzerland

Institute of Technology, Cambridge, Massachusetts 021 39, USA

versity of Iowa, 500 Newton Road, Iowa City, Iowa 52240, USA

09100 Cagliari, Sardinia

"J. AXELROD Laboratory of Clinical Science, Mental Health Intramural

D. ECCLESTON MRC Brain Metabolism Unit, University of Edinburgh,

D. FELIX Institut fur Hirnforschung der Universitat Zurich, August-Forel-

J. FERNSTROM Department of Nutrition and Food Science, Massachusetts

E. M. GAL Department of Psychiatry, State Psychopathic Hospital, The Uni-

G. L. GESSA Istituto di Farmacologia, UniversitA di Cagliari, Via Porcell 4,

* Unable to attend.

VIII PARTICIPANTS

J. GLOWINSKI ColEge de France, Laboratoire de Biologie MolCculaire, Groupe de Neuropharmacologie Biochimique, 1 1 place Marcelin-Berthelot, 75 Paris 5e, France

D. G. GRAHAME-SMITH MRC Clinical Pharmacology Unit, University Depart- ment of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE

s. KAUFMAN Laboratory of Neurochemistry, Mental Health Intramural Research Program, National Institute of Mental Health, 9000 Rockville Pike, Bethesda, Maryland 20014, USA

A. LAJTHA New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York 10035, USA

L. MATTRE Biological Research Laboratories, Pharmaceuticals Division, CIBA- GEIGY Limited, CH-4002 Basel, Switzerland

P. MANDEL Centre de Neurochimie, Centre National de la Recherche Scien- tifique, 11 rue Humann, 67085 Strasbourg Cedex, France

A. T. B. MOIR Scottish Home and Health Department, St Andrew’s House,

H. N. MUNRO Department of Nutrition and Food Science, Massachusetts

Edinburgh EHl 3DE

Institute of Technology, Cambridge, Massachusetts 02139, USA

S. s. OJA Department of Biomedicine, University of Tampere, Teiskontie 37, SF-33520 Tampere 52, Finland

Department of Biological Chemistry, University of California, School of Medicine, The Center for the Health Sciences, Los Angeles, California 90024, USA

M. SANDLER Bernhard Baron Memorial Research Laboratories, Department of Chemical Pathology, Queen Charlotte’s Maternity Hospital, Goldhawk Road, London W6 OXG

D. F. SHARMAN ARC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT

T. L. SOURKES Departments of Psychiatry and Biochemistry, McGill University,

S. ROBERTS

1033 Pine Avenue West, Montreal 112, Quebec, Canada

N. WEINER Department of Pharmacology, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220, USA

Editors: G. E. w. WOLSTENHOLME and DAVID w. FITZSIMONS

Editors’ note

As far as possible, we have followed the Recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature and the conventions of the Bio- chemical Journal (see Instructions to Authors). In view of the variety of liames in common use, we append a list of the trivial and systematic names of some compounds mentioned in the book.

dopa: 3-(3,4-dihydroxyphenyl)alanine (I) dopamine: 2-(3,4-dihydroxyphenyl)ethylamine, i.e. 4-(2-aminoethyl)benzene-

1,2-diol homogentisic acid: 2,5-dihydroxyphenylacetic acid homovanillic acid 4-hydroxy-3-methoxyphenylacetic acid 6-hydroxydopa : 3-(2,4,5-trihydroxyphenyl)alanine 5-hydroxyindoleacetic acid

(5HIAA): 5-hydroxy-3-indolylacetic acid (11) p-hydroxymandelic acid : hydroxy(4-hydroxypheny1)acetic acid 5-hydroxytryptamine

(5HT, serotonin); 5-hydroxytryptophol : a-ketoglutarate: melatonin: a-methyldopa : MK486 noradrenaline

NSD 1015 NSD 1055

(norepinephrine) :

octopamine: phenylethanolamine : pterin : tryptamine: tyramine : rn-tyramine:

2-(5-hydroxy-3-indolyl)ethylamine, i.e. 3-(2-aminoethyl)-5-indolol 2-(5-hydroxy-3-indolyl)ethanol 2-oxoglutarate N-acetyl-2-(5-methoxy-3-indolyl)ethylamine 3-(3,4-dihydroxyphenyl)-2-methylalanine ~-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid 2-amino-l-(3,4-dihydroxyphenyl)ethanol, i.e. 4-(2-amino-l-

3-hydroxybenzylhydrazine, i.e. 3-hydrazinomethylphenol 4-bromo-3-hydroxybenzyloxyamine, i.e. 5-aminooxy-3-bromo-

2-amino-l-(4-hydroxyphenyl)et hanol 2-amino-1-phenylethanol 2-amino-4-pteridinol 2-(3-indolyl)ethylamine 4-(2-aminoethyl)phenol 3-(2-aminoethyl)phenol

hydroxyethyl)benzene-1,2-diol

phenol

(I) CH2*CH(NHz)*COOH (I[)

H O ~ CH,.COOH 0.. OH H

Chairman’s introduction

R. J. WURTMAN

Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

It is no secret to this community that aromatic amino acids have a special significance in the functions of the brain. By ‘aromatic amino acids’ we are forced to restrict our attention to three such compounds : phenylalanine, tyrosine and tryptophan. (Tt is not that histidine is not important, but rather that the time at our disposal is not infinite.) These compounds are extremely important in normal brain function, in the pathophysiology of various disease states and in the responses of the brain to various drugs. Like the other amino acids present in dietary protein, they circulate in the blood and are taken up into brain, where they charge transfer RNAs and are subsequently incorporated into peptides and proteins. Furthermore, these amino acids can be hydroxylated within specific neurons, generating other amino acids which in turn are decarboxylated to yield biogenic monoamines that function as neurotransmitters. One can make an impressive list of brain diseases in which they participate, starting with phenyl- ketonuria, the prototypic inborn error of metabolism. Other brain diseases have, in recent years, been shown to respond favourably to treatment with hydroxylated amino acids not normally found in the circulation-L-dopa and L-5-hydroxytryptophan. The lack of adequate amounts of dietary protein (Shoemaker& Wurtman 1971) or the chronic consumption of proteins like corn that contain unfavourable proportions of these amino acids (Fernstrom & Wurtman 1971) can interfere with normal brain function and behaviour; these disturbances coincide with changes in the concentrations of the monoamine neurotransmitters in the brain and urine (Hoeldtke & Wurtman 1973).

Considering this basic recognition of the importance to brain of the aromatic amino acids, some of us thought that it might be useful to try to bring together representatives of the three communities of scientists who study these compounds. The first such community comprises those who are concerned with the factors that control the concentrations of these aromatic amino acids in the

2 R. J. WURTMAN

brain, their flux between brain and extracellular fluid, and their catabolism in brain. Amino acid transport into brain appears to be mediated by group-specific transport systems that differ markedly from, for example, the insulin-sensitive mechanisms that operate in skeletal muscle. As we shall see, phenylalanine, tyrosine and tryptophan can apparently be decarboxylated by brain enzymes to yield the corresponding simple amines ; they can also be transaminated, and the indole ring of tryptophan can be opened by an oxygenase, originally described from small intestine, that also cleaves D- and L-5-hydroxytryptophan, 5-hydroxytryptamine and melatonin (N-acetyl-5-methoxytryptamine) (Hirata & Hayaishi 1972).

The second group focuses on the use of these amino acids as precursors of the monoamine neutrotransmitters dopamine, noradrenaline and 5-hydroxytryptamine. Its concerns include the relationships between the neuronal concentrations of tyrosine or tryptophan and the rates at which these amino acids are hydroxylated to form dopa and 5-hydroxytryptophan respectively. Present information suggests the operation of two different mechanisms controlling the hydroxylations of tyrosine and tryptophan : the mechanism for tyrosine is thought to depend not on precursor availability but on the activity of a rate- limiting enzyme, tyrosine hydroxylase. As will be discussed, tyrosine hydroxylase activity may be controlled by end-product (i.e. catecholamine) inhibition and seems to increase or decrease when the physiological activity of ‘catechol- aminergic’ neurons changes. In contrast, it appears that the major factor controlling the rate at which brain neurons synthesize 5-hydroxytryptamine is neuronal concentration of tryptophan; hence, physiological actions such as eating which change this concentration thereby modify brain levels of 5-hydr- oxyindoles. In part of this symposium we shall consider the validity of these generalizations and the possible biological consequences of these two, very different, control systems.

The third community consists of those who are concerned with the varieties of proteins that the brain synthesizes, the loci of protein synthesis within neurons and other cells, and the extent to which the amino acid charging of brain tRNA and other fxtors control the rates at which specific proteins and proteins as a group are made. There is abundant evidence that the availability of amino acids, especially tryptophan, is of major importance in determining the overall rate of protein synthesisin another mammalian organ, the liver (Munro 1970). The extent to which hepatic messenger RNA can perform its function- the extent to which it aggregates with pairs of ribosomes, forming polysomes-is largely determined by the availability of amino acids. Tryptophan seems normally to be the limiting amino acid, probably because it is the least-abundant amino acid in most foods and in the protein reservoirs in our own tissues. One wonders whether brain

CHAIRMAN’S INTRODUCTION 3

protein synthesis might also be limited by tryptophan availability-especially within neurons that also use this scarce amino acid for the biosynthesis of 5-hydroxytryptamine.

These communities have tended to overlap in the pursuit of several specific problems that will be discussed here; for example, all three groups have used drugs like p-chlorophenylalanine, a-methyltryptophan and, more recently, L-dopa which affect the concentrations of amino acids in brain and also the rates of synthesis of both the monoamine neurotransmitters and proteins.

An emergent generalization is that the concentrations of precursors in mammalian cells may be crucial in controlling the rates of many reactions. Those of us who came into mammalology with a background of work on E.coli had learnt to believe that the quantities of enzyme proteins present in the cell, or the al- losteric state of these proteins, controlled the cell’s biochemical activity; thus genetics were the key to an understanding of physiology. Now we see more and more evidence that the availability of substrate-a nutritional factor-can limit enzymic reactions, at least in mammalian cells. An undercurrent throughout this symposium will be ‘brain nutrition’-the control of brain concentrations of three nutrients (phenylalanine, tyrosine and tryptophan) which it cannot provide for itself, and the extent to which these concentrations control the synthesis of compounds essential for normal brain function.

References

FERNSTROM, J. D. & WURTMAN, R. J. (1971) Effect of chronic corn consumption on serotonin content of rat brain. Nut. New Biol. 234, 62-64

HIRATA, F. & HAYAISHI, 0. (1972) New degradative routes of 5-hydroxytryptophan and serotonin by intestinal tryptophan 2,3-dioxygenase. Biochem. Biophys. Res. Commun. 47,

HOELDTKE, R. D. & WURTMAN, R. J. (1973) The excretion of catecholamines and catecholamine metabolites in kwashiorkor. Am. J. Clin. Nutr. 26, 205-210

MUNRO, H. N. (1970) A general survey of mechanisms regulating protein metabolism in mammals in Mammalian Protein Metabolism, vol. 4, pp. 3-130, Academic Press, New York

SHOEMAKER, W. J. & WURTMAN, R. J. (1971) Perinatal undernutrition: accumulation of catecholamines in rat brain. Science (Wash. D.C.) 171, 1017-1019

1 1 12-1 1 19

Control of plasma amino acid concentrations

H. N. MUNRO

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

Abstract The daily flux of amino acids in the body is extensive. About 300 g of protein is synthesized each day in an adult man. This requires the uptake and release of 150 g of essential amino acids, yet the minimum dietary requirement for essential amino acids is only 6 g while the customary diet contains 45 g. This indicates considerable and efficient recycling of the essential amino acids released by protein breakdown. Since plasma contains a total of 0.2 g of essential amino acids, recycling of amino acids between tissues will cause rapid turnover of the free amino acids in plasma.

Not all amino acid molecules released by the turnover of body proteins are transferred to the blood plasma. Considerable amounts can be reused for protein synthesis within cells; consequently, the equation of amino acid uptake by (or release from) tissues with the total flux of amino acids within the tissue is not valid. This difficulty can be overcome if an amino acid released by protein breakdown is not reused for synthesis. For example, some histidine molecules in actin and myosin of skeletal musclearemethylated after proteinsynthesis. The resulting 3-methylhistidine cannot be reutilized and is quantitatively excreted in the urine ; thus it is ameasureof muscle protein turnover. Recognitionofnon-reusable prod- ucts of protein degradation in other tissues would similarly be most useful.

After a meal containing protein, the liver monitors the access of the incoming amino acids to the systemic blood. For most essential amino acids, hepatic degradation is regulated in relation to adequacy of intake and rises sharply when intake exceeds requirements. In spite of this protection, the amount of a given essential amino acid in the systemic plasma increases progressively when its intake exceeds requirements. The dietary intake of an amino acid at which its plasma concentration starts to rise has been used to estimate the demand for that amino acid. Studies on young and old rats receiving various intakes of tryptophan show that the response of the amount of tryptophan in plasma to different dietary intakes of tryptophan varies with the age of the animal and the time of plasma sampling after meals. When the tryptophan intake exceeds the required amount, the activity of tryptophan oxygenase in liver displays a diurnal rhythm. In older animals, carbohydrate in the meal causes transfer of tryptophan from plasma to muscle, a phenomenon common to other amino acids.

H. N. MUNRO 6

The aim of this paper is to survey the factors regulating the plasma concentrations of amino acids, specifically, the aromatic amino acids. I shall begin by attempting to quantify roughly the daily flux of amino acids in the body of an adult man and in some individual tissues. This will demonstrate how extensive is the reutilization of amino acids released by protein turnover within the body; this factor has caused problems in the interpretation of some metabolic studies. I shall briefly describe the use of amino acids which are not reutilized to circum- vent this and shall then consider how amino acid metabolism adjusts to changes in protein intake so that plasma concentrations are maintained within tolerable limits. This regulation will be illustrated by reference to the use of various intakes of dietary tryptophan.

DAILY FLUX OF AMINO ACIDS IN THE BODY

The daily flux of amino acids in various compartments of the body of an adult man weighing 70 kg is depicted in Fig. 1. The quantities shown are derived from a variety of sources described elsewhere (Munro 1972a) and should be regarded as first approximations in an attempt to arrive at some conception of the mag- nitude of amino acid turnover in the body. Balance experiments show that the

PROTEIN INTAKE Minimum Usual

329 9 0 9

BODY PROTEIN : 10 OOOg

Muscle 75 (? 1 Plasma : Albumin 12

Secreted Fibrinogen 2

White blood cells 20

Haemoglobin 8

GUT 5 0 - 7 0 9

Absorbed FREE AMINO -17 70-150 g I

ACIDS

FAECAL N (as protein)

10 9

URINARY N (as protein 1 20-729

+ SKIN 29

FIG. 1. Diagram of daily amino acid flux in the body of a 70 kg man (see Munro 1972~).

CONTROL OF PLASMA AMINO ACIDS 7

average adult maintains nitrogen equilibrium on an intake of 32 g of high quality protein (over 40 g is required by people whose requirements exceed the average by two standard deviations). In contrast, the customary protein intake in Western countries is much higher; on average it is over 90 g daily. This intake joins about 70 g of protein secreted into the gastrointestinal tract as digestive juice, and especially as shed mucosal cells (Fauconneau & Michel 1970), so that the total daily load for absorption is commonly as high as 150 g. The pools of free amino acids in the tissues which receive this load contain at least 70 g and exchange readily and extensively with body protein. Experiments with I5N indicate that about 300 g of protein are synthesized daily in the body of an adult man. Fig. 1 shows estimates of the rates of synthesis of the proteins in some specific tissues. Some 200 g of body protein made daily are thus accounted for. The data for plasma proteins, haemoglobin and white cells are based on reasonably reliable estimates of daily turnover rates. The amount of protein secreted into the gastrointestinal tract is less reliable, because shed mucosal cells comprise most of the daily output of endogenous gut protein This protein loss is difficult to quantitate with adequate precision (Fauconneau & Michel 1970). The diagram also bears a figure for muscle protein turnover. This figure is based on the output of amino acids from muscle into the blood by fasting adults which, if continued for 24 h, would represent breakdown of 75 g of muscle (Cahilll972). While this rate of loss must be compensated after meals by a net uptake of amino acids by muscle in order to maintain equilibrium within the tissue, we shall see later that muscle protein turnover is not necessarily represented by the rate of amino acid output during fasting and may even be double the value shown in Fig. 1.

Some components in daily amino acid flu are expressed in Table 1 as quantities of essential and non-essential amino acids, including tryptophan, phenylalanine and tyrosine (Table 2). The average minimum amount of dietary protein required in order to maintain nitrogen equilibrium in the adult (32 g daily) need only contain 6 g of essential amino acids (Munro 19723). In contrast, of the 90 g of protein in the customary Western diet about half is present as essential amino acids. These 45 g join the essential amino acids entering the gut as endogenous proteins. Consequently, about 75 g of essential amino acids are absorbed daily. Compare this with the total amounts of essential and non- essential amino acids in the plasma, namely 0.2 and 0.5 g, respectively. SO it is to be expected that free amino acids will be rapidly transported out of the blood into the tissues. The tissues contain some 70 g of free amino acids, but four non-essential amino acids (glycine, glutamic acid, glutamine and alanine) represent 80 % of this, whereas only about 10 g of essential amino acids are present. Nevertheless, the daily turnover of 300 g of body protein requires incorporation

H. N. MUNRO 8

TABLE 1 Intake, tissue content and turnover of essential and non-essential amino acids for a 70 kg adult

Amino acid source Amino acids170 kg body wt

Total ( g ) Essential ( g ) Non-essential ( g ) -

Daily diet Minimum amino acid needs" 32 6 26 Western dietb 90 45 45 Absorbed (with secreted gut protein)b 150 75 75

Free amino acid pools' Plasma 0.7 0.2 0.5 Tissues 70 10 60

Daily body protein turnoverd 300 150 150

From Munro (19726). These data are taken from Fauconneau & Michel (1970). It is assumed that the mixed

proteins of the diet and the intestinal secretions contain about 50% essential amino acids, which is a reasonable estimate.

These are calculated for a 70 kg adult from data on free amino acid concentrations in the blood and tissues of the rat (Munro 1970). A few data on human tissues confirm their appli- cability.

From San Pietro & Rittenberg (1953), assuming that 50% of the amino acids incorporated into and released from body protein are essential amino acids.

TABLE 2 Intake, plasma content and turnover of tryptophan, phenylalanine and tyrosine for a 70 kg man

Amounts/70 kg man

- T ~ P fg) Phe (g) T Y ~ (g)

Daily diet Minimum needs" 0.2 -1.0- Western diet' 1 .o 4.2 3.1 Absorbed (with gut protein) 1.5 5.9 4.7

Plasma amino acid pool" 0.020 0.012 0.014 Daily protein turnoverd 3.3 13.0 10.5

a Requirement for nitrogen equilibrium (Munro 1972b) calculated for a 70 kg man. The requirement for phenylalanine includes the amount needed for tyrosine formation.

The average Western diet is taken to contain 90 g protein (Table l ) , with 60 g derived from animal sources and 30 g from vegetable sources. Based on the amino acid content of foods (FA0 Handbook 1970) and the proportions of these eaten, the approximate percentages of tryptophan, phenylalanine and tyrosine in food protein are respectively 1.2, 4.6 and 3.7 for animal foods and 1.0, 4.8 and 2.6 in vegetable foods. These percentages are not appreciably altered by variations in the major types of animal and vegetable protein sources used for the computations.

From data for human plasma (Munro 1969), assuming plasma volume to be 2% of body weight.

From amino acid content of carcass protein of mammals (Munro & Fleck 1969), and a daily protein turnover of 300 g (Table 1).


of 150 g of essential amino acids daily into tissue protein. Most of this must be derived from recycling of essential amino acids released by the tissues, since the diet customarily provides only about 45 g essential amino acids and nitrogen equilibrium can still be achieved when the total content of essential amino acids in the diet is as low as 6 g (Munro 1972b). This implies very efficient recycling of essential amino acids within the body.

A similar picture emerges when single essential amino acids such as tryptophan and phenylalanine and the semi-essential amino acid tyrosine are con- sidered (Table 2). The minimum dietary requirement for the first two are lg/day or less, whereas the daily turnover within the body is about 15-20 times the requirement. The amounts of these three amino acids i n the plasma are small compared with this daily turnover, about 1/150 for tryptophan and 1/1000 for both phenylalanine and tyrosine. This implies that the proportion of tryptophan in the plasma is unusually large in view of its function in body protein synthesis. These metabolic parameters computed for humans can be amplified by analogous calculations for the rat. Table 3 shows that the ratio of tryptophan to phenylalanine to tyrosine in the total free amino acid pool of the body and free in the tissues is about 1 : 4: 4 whereas in plasma it is 1 : 1.2: 1.4. If, instead of total plasma tryptophan, we compute the ratio of phenylalanine and tyrosine to non-albumin- bound tryptophan in plasma, the ratio becomes 1 : 4.8 : 5.8, as in the tissues. This implies that the bound tryptophan in plasma represents an excess (a reservoir) and that the tissues probably do not have a corresponding binding protein.

TABLE 3

Requirements, body protein content and free concentrations of tryptophan, phenylalanine and tyrosine in the rat

Amount (pmol/100 g body Ratios or tissue wt.)

Trp Phe TY r Trp Phe Tyr -

Daily requirements" 55 -450- 1 .o -9.0- Body protein content" 980 5 800 3 550 1.0 6.0 3.6 Body free amino acid" 2 9 8 1.0 4.5 4.0 Tissue free amino acidsb

Liver 5.6 21 25 1.0 3.8 4.5 Muscle 3.7 15 20 1.0 4.1 5.4 Brain 2.3 5.6 9.5 1.0 2.4 4.1 Plasma 6.5 7.7 9.4 1.0 1.2 1.4 Plasma (non-bound) (1.6) (1.0) (4.8) (5.8)

From Munro (1970). The daily requirements are those for rapid growth. From Williams et al. (1950), with the assumption that 25 % of plasma tryptophan is not

bound to albumin (Fernstrom et al., this volume, pp. 153-166).

10

AMINO ACID REUTILIZATION IN THE BODY

H. N. MUNRO

The preceding calculations constitute indirect evidence that the essential amino acids are efficiently reutilized. There is, however, much direct evidence of this phenomenon. If an amino acid such as [U-14C]arginine is injected into rats and the rate of loss of labelled arginine from liver protein is measured, the apparent half-life of mixed liver protein is 4.5 days, whereas if [gu~nidino-'~C]arginine is used, the half-life falls to 3.3 days (Arias et al. 1969). This is because [guanidino- 14C]arginine participates in the arginine-ornithine cycle for urea synthesis after release from liver protein and in the process the labelled guanidino-carbon atom of free arginine is replaced by 12C, thereby greatly reducing the reuse of the 14C label in protein synthesis. Thus the intracellular pool is made up of amino acids entering the tissue from the plasma and also of amino acids released by protein turnover. It has been estimated that recycling in the rat liver can account for 50% of the free amino acid pool in the fed state and 90% after a short fast (Can & Jeffay 1967).

The complexities of recycling on the interpretation of data obtained from analysis of free amino acid concentrations in plasma are illustrated by some recent studies of amino acid metabolism in skeletal muscle. By measuring arteriovenous differences in amino acid concentration in the forearms of human subjects, Cahill and his colleagues (Pozefsky et al. 1969; Marliss et al. 1971) were able to quantitate the amounts of individual amino acids added to or removed from the limb muscles as the blood passed through. After an overnight fast, there is a considerable net output of amino acids from the musculature equivalent to a daily loss of 75 g from the total muscle protein of the body (Cahilll972). Much of this released amino nitrogen is made up of alanine and glutamine, owing to transamination of the bulk of the amino acids released from muscle protein. The alanine and glutamine are quantitatively absorbed by the liver where both amino acids are donors of -NH2 for urea synthesis and where alanine also provides a source of carbon for gluconeogenesis (Fig. 2). When insulin is injected into fasting subjects, the release of amino nitrogen into the bloodstream is sharply reduced. Pozefsky et al. (1969) consider this to be due to a reduction in rate of degradation of muscle proteins. However, it could also happen if the well known stimulation of muscle protein synthesis by insulin results in more rapid removal of amino acids from the intracellular pool, so that less is available for release into the blood (Fig. 2). These hypotheses could be tested directly if an amino acid that is not reutilized for protein synthesis is released from muscle breakdown. We have identified such an amino acid- 3-methylhistidine (Young et al. 1972). This is present in both the actin and myosin of muscle and is produced by methylation of histidine after the peptide

CONTROL OF PLASMA AMINO ACIDS I1

PLASMA t BLOOD PROTEINS A M I N O A C 5

p h u L FIG. 2. Recycling of amino acids within skeletal muscle and between muscle and liver (mainly as alanine and glutamine).

chains of these proteins have been made (Fig. 3). Injected 3-methylhistidine is metabolized by the rat to N-acetyl-3-methylhistidine, and the free and con- jugated 3-methylhistidine are then rapidly and quantitatively excreted in the urine. Thus, release of 3-methylhistidine from muscle into the blood and its excretion in urine provide an absolute measure of the rate of degradation of muscle protein, provided that other tissues are negligible sources and that the diet is free from sources of 3-methylhistidine such as meat. Recently, for example, we measured the urinary excretion of 3-methylhistidine by three obese patients on a 20-day fast (Young et al. 1973). Over this period, total body protein content fell only 10-15 % whereas urinary output of 3-methylhistidine nitrogen declined by about 40 %. Thus, the catabolism of muscle protein must

\ SY NTHE.S 1 S' CH3- METH I ON I NE \ I

HI

MYOSIN

RECYCLl ~~

\ 34eh I STI DINE

FIG. 3. The use of 3-methylhistidine in measuring rate of muscle protein breakdown without reutilization of amino acids.

12 H. N. MUNRO

be considerably reduced during fasting, through adaptation of protein breakdown. Other non-reusable amino acids would be most useful in evaluating changes in amino acid metabolism. It should be noted that calculations based on 3-methylhistidine content of human muscle suggest that the amount excreted is derived from breakdown of 150 g of muscle protein daily, more than twice thie figure calculated from arteriovenous differences by Pozefsky et al. (1969). This implies extensive recycling within the muscle cell of the amino acids released by turnover.

The interpretation of amino acid exchanges between tissues and blood has also been attempted for the human brain. Felig et al. (1973) have measured the arteriovenous difference in the brain for 15 amino acids including six essential amino acids in fasting adults and observed uptake of all amino acids by the brain. On the assumption of a blood flow of 1.2 I/min (Lewis et al. 1960), this means that about 30 g of amino acids are taken up every 24 h. It is difficult to interpret such data, since they imply either that an equivalent quantity of amino nitrogen is released from the brain at other times of day, notably after mealls, or else that the uptake is balanced by a loss into blood not drained by the jugular vein, such as the spinal cord and its cerebrospinal fluid. In this connection, Aoki et al. (1972) have found that red cells participate extensively as donors of glutamate to muscle. In future, arteriovenous studies may need to include red cells as well as plasma before a balance sheet of uptake and release of amino acids can be drawn.

THE LIVER AND REGULATION OF FREE AMINO ACID CONCENTRATIONS'

One reason for the limited requirement for essential amino acids is that the key catabolic enzymes for seven of them are restricted to the liver (Miller 1962). Consequently, essential amino acids liberated by protein breakdown within other tissues such as muscle will be reutilized with considerable efficiency if these amino acids do not leak into the blood in large amounts and reach the liver. It is, therefore, not surprising that the liver functionsespecially in monitoring the intake of essential amino acids and catabolizing the excess over requirements, whereas muscle liberates mainly non-essential amino acids for transport to the liver.

Although the recycling of amino acids within the body is extensive compared with requirements (Table l), the mechanisms for detecting and dealing with amounts of dietary amino acids in excess of needs are nevertheless surprisingly precise. The liver provides the major site of regulation. Amino acids absorbed after a meal rich in protein can raise the amounts of free amino acids in the portal vein considerably during the absorptive period, whereas their concentra-

CONTROL OF PLASMA AMIKO ACIDS 13

tions in the systemic circulation change much less. Studies on dogs bearing cannulae in the blood supply to and from the liver show that this is due to the enormous capacity of the liver to remove amino acids from the portal blood and thus to regulate their flow into the systemic circulation (Elwyn 1970). Conse- quently, amino acid metabolism after meals causes diurnal rhythms in liver protein metabolism which affect both the rate of synthesis of liver proteins and the amounts of enzymes involved in the catabolism of amino acids (Fishman et al. 1969).

As a result of these adaptive responses by the liver to meals, the systemic circulation is protected against excessive changes in the amounts of free amino acids entering the body. There is reason to believe that, in the case of the essential amino acids, this catabolic response is finely adjusted to the amount entering the body in relation to requirements. For example, Brookes et al. (1972) added successively greater amounts of [14C]lysine to the diet of growing rats; the amounts of the other dietary amino acids were kept constant. Consumption of amounts of total dietary [ 14C]lysine below requirements led to a constant low release of 14CQ,. However, when lysine intake was raised above the requirement for optimal growth, the proportion of [14C]lysine released as 14C0, rose steeplyand further addition of lysine to the diet increased 14CQ, production still more. Consequently, there was a sharp inflection in 14C02 output at the point where lysine intake was just sufficient to support maximal growth rate. This pattern of response took several days to develop, presumably because feedback of information from the body as a whole to the liver, where lysine is degraded, required this period to establish increased hepatic concentrations of free lysine.

RESPONSE OF PROTEIN METABOLISM TO DIFFERENT INTAKES OF TRYPTOPHAN

Although the liver is thus equipped to respond sensitively to intakes of amino acids in excess of requirements, some of the extra amino acid enters the systemic blood. Consequently, the sudden increase in hepatic oxidation of an essential amino acid at the point at which requirements are met usually coincides with an increase in the blood concentration of the amino acid. This may be the signal from the tissues required by the liver to turn on the catabolic process. Recently, we have examined several aspects of tryptophan metabolism in rats with different intakes using both young rats (54 g initial weight) with a high requirement and older rats (300-400 g) with a lower requirement for tryptophan (Young & Munro 1973). Groups of rats from each age group were given diets providing amino acids in place of protein, and the intake of tryptophan was varied from 0 to 0.33% of the diet. The requirement for tryptophan in the young rat has

14

c H. N. MUNRO

4

Requirement

1 Old R a t s

2300 h

1100 h

I

0 0. I 0.2 0.3 T r y p t o p h a n in diet ( O / O )

FIG. 4. Concentrations of tryptophan in the plasma of young and mature rats fed amino acid diets containing various amounts of tryptophan, other nutrients being adequate and constant. The arrows indicate requirements for tryptophan reported in the literature for weanling and adult rats (Munro & Young, unpublished data).

been taken as 0.1 1 % (Rama Rao et al. 1959) and for mature rats as 0.03 % (Smith & Johnson 1967) (see Fig. 4). Plasma tryptophan was measured twice: a day (at 1100 and 2300) after nine days on these diets. In young rats, the plasma tryptophan concentration increased at both these times as soon as the requirement had been met (Fig. 4). In the mature animals, the increase was evident for blood samples at 2300, the rat's habitual time of feeding, whereas the results obtained at 1100 did not provide this clear evidence of a requirement-related increment. Thus, blood amino acid responses can be used more sensitively during the absorptive phase as monitors of requirements. It would be interesting to know whether changing the proportion of plasma tryptophan bound to albumin by altering the amount of non-essential fatty acid in the plasma (see


Fernstrom et al., this volume, pp. 153-166) results in a change of the plasma tryptophan response curve, thus indicating an effect of albumin binding on the use of tryptophan.

From Fig. 4 it can be seen that the older rats had a much lower plasma tryptophan concentration at 2300 than at 1100 for all except the greatest intakes of tryptophan. A constituent of the food which the rat begins to eat just before 2300 must have caused this reduction. We know from other work that dietary carbohydrate decreases the amounts of free amino acids in the plasma owing to the insulin-dependent deposition of the amino acids in muscle. Therefore, we measured the concentrations of free tryptophan in muscle and showed that the reduction in plasma concentration did correlate with a rise in muscle concentration at 2300 (Table 4). This presumably means that the carbohydrate content of the meal caused transfer of tryptophan into muscle irrespective of the tryptophan content of the diet. If the diet is inadequate in tryptophan, this will presumably limit tryptophan supply to other tissues even more-a feature of carbohydrate action without adequate dietary amino acid supply which I have pointed out previously (Munro 1964). It is clear from Fig. 4 that, unlike the older rats, the young rats did not show a reduction in the amount of plasma tryptophan when they consumed meals containing little or no tryptophan. This correlates with Fernstrom’s observation (personal communication) that carbohydrate ad- ministration affects plasma tryptophan concentrations differently in young and mature rats.

We also measured the activity of tryptophan oxygenase in the liver and observed that activity rose after meals (from observations at 2300) only in those animals that were receiving more than their dietary requirements of tryptophan (Table 4). This indicates that the enzyme is sensitive to the amount of dietary

TABLE 4

Effect of dietary tryptophan concentration on tryptophan content of plasma and muscle, and on activity of tryptophan oxygenase in older rats

2300 1100 2300 I100

Tryptophan oxygenase (units of activitylg liver)

2300 I100

0.000 3.2 6.6 0.016 4.3 8.5 0.033 4.1 8.0 0.066 5.2 8.2 0.108 6.2 10.9 0.220 12.2 10.5

17.5 14.3 17.0 14.2 18.0 15.6 16.3 12.3 17.0 12.3 17.4 13.8

2.0 1.1 2.5 1.8 2.1 1.8 3.2 1.6 3.2 1.5 3.2 1.4

Data are from Young & Munro (1973).

16 H. N. MUNRC)

T

I L

C--.. 0.108Y0Trp

0 0.01 6 %Trp O----

10 1800 0200 2200

Time of day(hours1 FIG. 5. Hepatic activity of tryptophan oxygenase, as shown by production of kynurenine, at different times for mature rats fed small or large amounts of tryptophan; other dietary factors are adequate and constant (Young & Munro 1973).

tryptophan with respect to requirements, and that it exercises this monitoring action at the time of absorption of the meal. We verified this conclusion by feeding rats large and small amounts of dietary tryptophan and observinig diurnal changes in tryptophan oxygenase due to variations in food intake. Fig. 5 shows that the superoptimal intake induced a meal-related increase in enzyme activity that was absent when suboptimal amounts of tryptophan were fed.

ACKNOWLEDGEMENT

The original data reported here were supported by USPHS grant AM 15364.

References

AOKI, T. T., BRENNAN, M. F., MULLER, W. A., MOORE, F. D. & CAHILL, G . F. (1972) Effect of insulin on muscle glutamate uptake. Whole blood versus plasma glutamate analysis. J. Clin. Invest. 51, 2889-2894


ARIAS, I. M., DOYLE, D. & SCHIMKE, R. T. (1969) Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J . Bid. Chem. 244, 3303-3315

BKOOKES, I. M., OWEN, F. N. & GARRIGUS, U. S. (1972) Influence of amino acid level in the diet upon amino acid oxidation by the rat. J. Nutr. 102, 27-34

CAHILL, G . F. (1972) Carbohydrates in Symposium on Total Parenteral Nutrition (Vanamee, P. & Shils, M. E., eds.), pp. 45-51, American Medical Association, Chicago

ELWYN, D. (1970) The role of the liver in regulation of amino acid and protein metabolism in Mammalian Protein Metabolism, vol. 4 (Munro, H. N., ed.), pp. 523-571, Academic Press, New York

FAUCONNEAU, G. & MICHEL, M. C. (1970) The role of the gastrointestinal tract in the regulation of protein metabolism in Mammalian Protein Metabolism, vol. 4 (Munro, H. N., ed.), pp. 481-522, Academic Press, New York

FELIG, P. & WAHREN, J. (1971) Amino acid metabolism in exercising man. J. Clin. Iizvest. 50,

FELIG, P., WAHREN, J. & AHLBORG, G. (1973) Uptake of individual amino acids by the human brain. Proc. Soc. Exp. Biol. Med. 142, 230-231

FISHMAN, B., WURTMAN, R. J. & MUNRO, H. N. (1969) Daily rhythms in hepatic polysome profiles and tyrosine transaminase: role of dietary protein. Proc. Natl. Acad. Sci. U.S.A.

CAN, J. C. & JEYYAY, H. (1967) Origin and metabolism of the intracellular amino acid pools in rat liver and muscle. Biochim. Biophys. Acta. 198, 448459

LEWIS, B. M., SOKOLOFF, L., WECHSLER, R. L., WENTZ, W. B. & KETY, S. S. (1960) A method for the continuous measurement of cerebral blood flow in man by means of radioactive krypton. J . Clin. Invest. 39, 707-716

MARLISS, E., AOKI, T. T., POZEFSKY, T., MOST, A. & CAHILL, G. F. (1971) Muscle and splanch- nic glutamine and glutamate metabolism in post absorptive and starved man. J . Clin. Invest. 50, 814-817

MILLER, L. L. (1962) The role of the liver and the non-hepatic tissues in the regulation of free amino acids levels in the blood in Amino Acid Pools (Holden, J. T., ed.), pp. 708-721, Elsevier, Amsterdam

MUNRO, H. N. (1964) General aspects of the regulation of protein metabolism by diet and hormones in Mammalian Protein Metabolism, vol. 1 (Munro, H. N. & Allison, J. B., eds.), pp. 381481, Academic Press, New York

MUNRO, H. N. (1969) in Mammalian Protein Metabolism, vol. 3 (Munro, H. N., ed.), pp. 113- 182, Academic Press, New York

MUNRO, H. N. (1970) Free amino acid pools and their role in regulation in Mammalian Prorein Metabolism, vol. 2 (Munro, H. N., ed.), pp. 286-299, Academic Press, New York

MUNRO, H. N. (1972~) Basic concepts in the use of amino acids and protein hydrolysates for parenteral nutrition in Symposium on Total Parenteral Nutrition (Vanamee, P. & Shils, M. E., eds.), pp. 7-35, American Medical Association, Chicago

MUNRO, H. N. (19726) Amino acid requirements and metabolism and their relevance to parenteral nutrition in Parenteral Nutrition (Wilkinson, A., ed.), pp. 34-67, Churchill, Livingstone, Edinburgh & London

MUNRO, H. N. & FLECK, A. (1969) Analysis of tissues and body fluids for nitrogenous con- stituents in Mammalian Protein Metabolism, vol. 3 (Munro, H. N., ed.), pp. 423-525, Academic Press, New York

POZEFSKY, T., FELIG, P., TOBIN, J., SOELDNER, J. S. & CAHILL, G. F. (1969) Amino acid balance across the tissues of the forearm in post-absorptive man: effects of insulin at two dose levels. J . Clin. Invest. 48, 2273-2280

RAMA RAO, P. B., METTA, V. C. &JOHNSON, B. C. (1959) The amino acid composition and the nutritive value of proteins. I. Essential amino acid requirements of the growing rat. J . Nutr. 69, 387-391

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Discussion

Fernstrom: Does Fig. 5 show the amount of kynurenine produced in vitro? Munro: Yes. Those results give us an index of the total amount of active

enzyme because it was assayed in the presence of cofactors. It does not indicate the amount of substrate passage in the whole animal. Obviously, substrate concentrations are important in relation to enzymes as well as the total amount of enzymes present.

Fernstrom: When we fed rats diets containing carbohydrate, or carbohydrate plus protein, we found that whereas tyrosine aminotransferase is rapidly activated by protein consumption tryptophan oxygenase is not. Nonetheless, we noticed that the oxygenase activity reaches a peak around the time of day that the animal eats, so that while the enzyme may not be activated by tryptophan it seems to be active at an appropriate time of day, that is, when large amounts of tryptophan are entering the portal vein from the gut.

Munro: This was not apparent when we studied animals receiving low intakes of tryptophan. The adaptation of lysine oxidation to excessive intake indicates that equilibration takes about ten days (Brookes et al. 1972). So, the information demanding these responses must be somehow relayed to the liver. The minimum requirement in the adult human of 6 g total essential amino acids (cf. p. 7 and Table 1) is able to prime the 150 g cycle each day. The evidence regarding the sensitivity of enzymes degrading amino acids to appropriate loads will depend on the animal preparation used.

Grahame-Smith: For a long time I have tried to understand the correlation between activity of regulating enzymes (tryptophan oxygenase, tyrosine aminotransferase) with the activity of the enzymes found in liver homogenates iul viiro.

aromatic amino acids in the brain...in normal brain function, in the pathophysiology of various...

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